Pulmonary surfactant protein and related polypeptide

ABSTRACT

The present invention relates to a human SP18 monomer protein-related polypeptide useful in forming a synthetic pulmonary surfactant. The present invention also relates to a method of treating neonatal respiratory distress syndrome comprising adminstering a therapeutically effective amount of synthetic pulmonary of the present invention. Further contemplated by the present invention is a composition containing human SP18 monomer and human SP18 dimer but no other pulmonary surfactant proteins. A recombinant DNA molecule capable of expressing, without post-translational proteolytic processing, mature human SP18 monomer, and methods of using the recombinant DNA molecule are also contemplated.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation-in-part application of copending application Ser.No. 141,200, filed Jan. 6, 1988, now abandoned, entitled PulmonarySurfactant Protein and Related Polypeptide (Cochrane, et al.), whichapplication is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to SP18 monomer-related polypeptidesuseful in forming synthetic pulmonary surfactants. The present inventionalso relates to a recombinant nucleic acid molecule carrying astructural gene that encodes human SP18 monomer protein and the use ofsuch a recombinant molecule to produce human SP18 monomer.

BACKGROUND

Pulmonary surfactant (PS) lines the alveolar epithelium of maturemammalian lungs. Natural PS has been described as a "lipoproteincomplex" because it contains both phospholipids and apoproteins thatinteract to reduce surface tension at the lung air-liquid interface.

Since the discovery of pulmonary surfactant, and the subsequent findingthat its deficiency was the primary cause of neonatal respiratorydistress syndrome (RDS), a number of studies have been directed towardsdeveloping effective surfactant replacement therapy for affectedindividuals, particularly infants, using exogenous PS. For instance,improvements in lung function as measured by a decrease in mean airwaypressure and oxygen requirements have been demonstrated using exogenoussurfactants in human pre-term infants. See Hallman, et al., Pediatrics,71:473-482 (1983); Merritt, et al., J Pediatr., 108:741-745 (1986);Hallman, et al., J. Pediatr., 106:963-969 (1985); Morley, et al.,Lancet, i:64-68 (1981); Merritt, et al., New England J. Med.,315:785-790 (1986), Smyth, et al., Pediatrics, 71:913-917 (1983);Enhorning, et al., Pediatrics, 76:145-153 (1985); Fujiwara, et al., TheLancet, 1:55-59 (1980); Kwong, et al., Pediatrics, 76:585-592 (1985);Shapiro, et al., Pediatrics, 76:593-599 (1985); Fujiwara, in "PulmonarySurfactant", Robertson, B., Van Golde, L. M. G., Batenburg J. (eds),Elsevier Science Publishers, Amsterdam, pp. 479-503, (1984).

From a pharmacologic point of view, the optimal exogenous PS to use inthe treatment of RDS would be one completely synthesized in thelaboratory, under controlled and sterile conditions, with negligiblebatch-to-batch variability in properties. To minimize the possibility ofimmunologic complications, the apoprotein component of an exogenous PSshould be identical to that found in humans. Unfortunately, thecomposition of naturally occurring PS is complex, and the art has notyet identified all of the biochemical components that generate thebiophysical properties needed for high physiologic activity in lungs. Inparticular, the art has failed to characterize all of the apoproteinspresent in natural PS or identify the function of the PS apoproteinspresently known.

It should be noted that the literature on PS apoproteins and their rolesin surfactant function is complex, inconsistent and sometimescontradictory because heterogeneous apoprotein preparations were used inmany studies. To data, the art has not definitively established thenumber of different apoproteins present in natural PS.

Of particular interest to the present invention is the use of a lowmolecular weight (LMW) human PS-associated apoprotein as a component inan exogenous surfactant. Several studies have attempted to isolated ordefine human PS LMW apoproteins using biochemical techniques. See, forexample, Phizackerley, et al., Biochem. J., 183:731-735 (1979), Revak,et al., Am. Rev. Resp. Dis., 134:1258-1265 (1986), Suzuki et al., Eur.J. Respir. Dis., 69:335-345 (1986), Taeusch, et al., Pediatrics,77:574-581 (1986), Yu, et al., Biochem. J., 235:85-89 (1986), Whitsett,et al., Pediatric Res., 20:460-467 (1986), Whitsett, et al., PediatricRes., 20:744-749 (1986), Takahashi, et al., Biochem. Biophys. Res.Comm., 135:527-532 (1986), Suzuki et al., Exp. Lung. Res., 11:61-73(1986), Curstedt, et al., Eur. J. Biochem., 168:255-262 (1987), Notter,et al., Chem. Phys. Lipids, 44:1-17 (1987), and Phelps, et al., Am. Rev.Respir. Dis., 135:1112-1117 (1987).

Recently, the art has begun to apply the methods of recombinant DNAtechnology to overcome the problems associated with not being able toisolate to homogeneity the individual LMW PS apoproteins. For instance,Glasser, et al., Proc. Natl. Acad. Sci., U.S.A., 84:4007-4011 (1987)reported a cDNA derived sequence of amino acid residues that forms atleast a portion of human precursor protein from which at least onemature LMW apoprotein, which they designated SPL(Phe), is formed. WhileGlasser, et al. were not able to determine the carboxy-terminal residueof SPL(Phe), and therefore were not able to identify its completesequence, they did predict that mature SPL(Phe) was about 60 amino acidsin length.

Jacobs, et al, J. Biol. Chem., 262:9808-9811 (1987) have described acDNA and derived amino acid residue sequence for a human precursorprotein similar to that described by Glasser, et al., supra. However,according to Jacobs et al. the mature LMW apoprotein, which theydesignated PSP-B, formed from the precursor would be 76 amino acidresidues in length. In addition, Jacobs, et al., noted that it was notclear that any PS apoprotein derived from the reported precursor proteinwas present in the surfactant preparations that had been studiedclinically by others.

From the foregoing it can be seen that the literature contains multiplenomenclature for what is apparently the same PS apoprotein. Therefore,for ease of discussion, the mature apoprotein derived from the precursorprotein described by Glasser, et al., supra, and Jacobs, et al., supra,will be referred to herein generally as "SP18", with the monomeric anddimeric forms being referred to as "SP18 monomer" and "SP18 dimer",respectively, when appropriate.

The canine SP18 precursor has been described by Hawgood, et al., Proc.Natl. Acad. Sci. U.S.A., 84:66-70 (1987) and Schilling, et al.,International Patent Application WO 86/03408. However, it should benoted that both these studies suffered the same inability to define themature, biologically active form of SP18 to the Glasser, et al., supra,and Jacobs, et al., supra, studies.

Warr, et al., Proc. Natl. Acad. Sci., U.S.A., 84:7915-7919 (1987)describe a cDNA derived sequence of 197 amino acid residues that forms aprecursor protein from which a mature LMW apoprotein, they designate asSP5, is formed. Like the studies attempting to describe SP18, Warr, etal., were unable to determine the carboxy terminal residue of the matureprotein formed from the precursor protein sequence, and thus were notable to definitively characterize SP5.

Because the amino acid residue sequence of the precursor proteinreported by Warr, et al., is different from that reported by Glasser, etal., and Jacobs, et al., it therefore appears that the art hasdetermined that natural PS contains at least two LMW apoproteins.However, the biologically active forms of those proteins has remainedundetermined.

SUMMARY

It has now been found that human SP18 is a homodimeric protein (SP18dimer) whose mature subunit protein (SP18 monomer) displays an apparentmolecular weight of about 9,000 daltons when determined by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

It has also been discovered that human SP18 can function as an activeingredient in a synthetic pulmonary surfactant in the absence of otherpreviously identified pulmonary surfactant proteins.

In addition, the carboxy-terminal amino acid residue sequence of themature human SP18 monomer protein has been determined, and thus itsnaturally occurring form has now been discovered.

Thus, the present invention contemplates a composition comprisingsubstantially isolated or substantially pure human SP18 monomer.

Also contemplated by the present invention is a DNA segment consistingessentially of a DNA sequence encoding human SP18 monomer protein.

Another embodiment of the present invention is a recombinant nucleicacid molecule comprising a vector operatively linked to a structuralgene capable of expressing, without post-translational proteolyticmodification, human SP18 monomer protein.

Also contemplated is a method of preparing human SP18 monomer proteincomprising:

(a) initiating a culture, in a nutrient medium, of mammalian cellstransformed with a recombinant DNA molecule comprising a vectoroperatively linked to a structural gene capable of expressing, withoutpost-translational proteolytic modification, human SP18 monomer;

(b) maintaining said culture for a time period sufficient for said cellsto express human SP18 monomer protein from said recombinant DNAmolecule; and

(c) recovering said protein from said culture.

Further contemplated is a polypeptide consisting essentially of at least10 amino acid residues and no more than about 60 amino acid residuescorresponding in sequence to the amino acid residue sequence of humanSP18 monomer, said polypeptide, when admixed with a pharmaceuticallyacceptable phospholipid form a synthetic surfactant having a surfactantactivity greater than the surfactant activity of the phospholipid alone.

In another embodiment, the subject invention contemplates a syntheticpulmonary surfactant comprising a pharmaceutically acceptablephospholipid admixed with a polypeptide consisting essentially of atleast 10 amino acid residues and no more than about 60 amino acidresidues corresponding in sequence to the amino acid residue sequence ofhuman SP18 monomer.

A further embodiment of the subject invention is a method of treatingrespiratory distress syndrome comprising administering a therapeuticallyeffective amount of a synthetic pulmonary surfactant, said surfactantcomprising a pharmaceutically acceptable phospholipid admixed with aneffective amount of a polypeptide consisting essentially of at least 10amino acid residues corresponding in sequence to the amino acid residuesequence of human SP18 monomer, said polypeptide, when admixed with apharmaceutically acceptable phospholipid forms a synthetic surfactanthaving a surfactant activity greater than the surfactant activity of thephospholipid alone.

Another embodiment of the subject invention is a method of treatingrespiratory distress syndrome comprising administering a therapeuticallyeffective amount of a synthetic pulmonary surfactant, said surfactantcomprising an effective amount of either substantially isolated humanSP18 monomer or substantially pure human SP18 monomer admixed with apharmaceutically acceptable phospholipid.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a 750 nucleotide cDNA sequence (top lines) anddeduced amino acid residue sequence (bottom lines). The number to theright of each line of nucleotides represents the numerical position inthe sequence of the nucleotide at the end of each line. The nucleotidesare grouped into codons, 15 codons per line, with the amino acid residuecoded for by each codon shown in triple letter code directly below thecodon. The numerical position of some residues in the amino acid residuesequence encoded by the cDNA is shown below the residues. Theamino-terminal amino acid residue of mature human SP18 monomer is Phe(encoded by nucleotides 187-189) and is designated residue number 1. Thecarboxy-terminal amino acid residue is Asp at residue position 81(encoded by nucleotides 427-429). A structural gene encoding mature SP18monomer therefore contains 81 codons and has a nucleotide sequence thatcorresponds to nucleotides 187-429.

FIG. 2 illustrates the protein elution profile of PS apoproteins from aBio-Sil HA (silicic acid) column. Results of Pierce BCA protein assay(solid line) and phospholipid analyses (broken line) are shown forselected fractions. Two milliliters (ml) were collected per fraction.Positive protein assay in fractions 28 to 33 is due to the presence ofphospholipids.

FIG. 3 illustrates a Silver-stained SDS-PAGE of low molecular weight(LMW) PS apoproteins. Lanes A and D show a sample after silicic acid orSephadex LH-20 chromatography; both LMW proteins are present. Lanes B,C, E and F show the resolution of SP18 (lanes B and E) and SP9 (lanes Cand F) following chromatography on Sephadex LH-60. Molecular weightstandards are shown in lane G. Lanes A-C are unreduced samples, lanesD-F contain identical samples reduced with β-mercaptoethanol prior toelectrophoresis.

FIGS. 4A and 4B illustrate inflation and deflation pressure/volumecurves of fetal rabbit lungs 30 min after intratracheal instillation of100 ul of saline (open circles), 2 mg phospholipides (PL) DPPC:PG, 3:1(closed circles), PL+10 ug SP9 (open squares) PL+10 ug SP18 (closedsquares), or 2 mg natural human surfactant (closed triangles). Data areexpressed as the mean of 4 animals±one standard deviation.

FIGS. 5A, 5B, 5C, and 5D illustrate fetal rabbit lung tissue samples(×125 magnification, hematoxylin-eosin stain) following treatment withsaline (A), natural human surfactant (B), phospholipids DPPC:PG (C) orphospholipids plus LMW apoproteins (SP9+SP18) (D).

FIG. 6 illustrates the surfactant activity of exemplary polypeptidecontaining synthetic surfactants of the present invention. Surfactantactivity was determined by measuring the pressure gradient across anair/liquid interface using the pulsating bubble technique. The pressuregradient (ΔP) across the surface of the bubble is the absolute value ofthe pressure recorded in centimeters of H₂ O. The results obtained foreach synthetic pulmonary surfactant are identified by the polypeptide inthe surfactant. Results obtain for surfactants consisting ofphospholipid alone (i.e., with no peptide or protein admixed therewith)are identified as PL. The results obtained using a control peptidehaving only 8 amino acid residues and having a sequence corresponding tohuman SP18 monomer residues 74-81 (p74-81) are also shown. The data timepoints shown were obtained at 15 seconds, 1 minute and 5 minutes.

FIGS. 7A and 7B are two graphs that illustrate the results of a staticcompliance study of exemplary synthetic surfactants of this inventionusing the fetal rabbit model previously described in Revak, et al., Am.Rev. Respir. Dis., 134:1258-1265 (1986). Following instillation of asynthetic surfactant or control into the trachea, the rabbit wasventilated for 30 minutes prior to making static compliancemeasurements. The "x" axis represents the pressure in cm of water, whilethe "y" axis represents the volume in ml/kg of body weight. The graph onthe left represents values at inflation and the graph on the rightrepresents deflation values. The results for the following testedsurfactants are illustrated: natural surfactant (open square with a dotin the center), phospholipid (PL) with 7% p52-81 (a polypeptidecorresponding to residues 52 to 81 of SP18) (closed diamonds); PL with3% P52-81 (closed squares with white dot in center); PL with 7% p36-81(open diamonds); PL with 3% p66-81 (closed squares); PL with 3% p1-15(open squares) and PL control (closed triangles).

FIGS. 8A and 8B are two graphs that illustrate the results of a staticcompliance study of exemplary synthetic surfactants of the invention.The procedure was performed as described in FIG. 7 except that adifferent instillation procedure was used. The "x" and "y" axis andright and left graphs are as described in FIG. 7. The results for thefollowing tested surfactants are illustrated: natural surfactant (opensquares with a dot in the center); phospholipid (PL) with 10% p51-81(closed diamonds); PL with 10% p51-76 (closed squares); and PL (closedtriangles).

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Amino Acid: All amino acid residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature, J.Biol. Chem., 243:3557-59, (1969), abbreviations for amino acid residuesare as shown in the following Table of Correspondence:

                  TABLE OF CORRESPONDENCE                                         ______________________________________                                        SYMBOL                                                                        1-Letter      3-Letter    AMINO ACID                                          ______________________________________                                        Y             Tyr         L-tyrosine                                          G             Gly         glycine                                             F             Phe         L-phenylalanine                                     M             Met         L-methionine                                        A             Ala         L-alanine                                           S             Ser         L-serine                                            I             Ile         L-isoleuine                                         L             Leu         L-leucine                                           T             Thr         L-threonine                                         V             Val         L-valine                                            P             Pro         L-proline                                           K             Lys         L-lysine                                            H             His         L-histidine                                         Q             Gln         L-glutamine                                         E             Glu         L-glutamic acid                                     W             Trp         L-tryptohan                                         R             Arg         L-arginine                                          D             Asp         L-aspartic acid                                     N             Asn         L-asparagine                                        C             Cys         L-cysteine                                          ______________________________________                                    

It should be noted that all amino acid residue sequences are representedherein by formulae whose left to right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a bond to a radical such as Hand OH (hydrogen and hydroxyl) at the amino- and carboxy-termini,respectively, or a further sequence of one or more amino acid residues.In addition, it should be noted that a virgule (/) at the right-hand endof a residue sequence indicates that the sequence is continued on thenext line.

Polypeptide and Peptide: Polypeptide and peptide are terms usedinterchangeably herein to designate a linear series of no more thanabout 60 amino acid residues connected one to the other by peptide bondsbetween the alpha-amino and carboxy groups of adjacent residues.

Protein: Protein is a term used herein to designate a linear series ofgreater than about 60 amino acid residues connected one to the other asin a polypeptide.

Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar moiety(pentose), a phosphate, and a nitrogenous heterocyclic base. The base islinked to the sugar moiety via the glycosidic carbon (1' carbon of thepentose) and that combination of base and sugar is a nucleoside. Whenthe nucleoside contains a phosphate group bonded to the 3' or 5'position of the pentose it is referred to as a nucleotide.

Base Pair (bp): A partnership of adrenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a double stranded DNA molecule.

B. SP18 Monomer-Containing Compositions

The present invention contemplates a SP18 monomer-containing composition(subject protein composition) wherein the SP18 monomer is present ineither substantially isolated or substantially pure form. By "isolated"is meant that SP18 monomer and SP18 dimer are present as part of acomposition free of other alveolar surfactant proteins.

By "substantially pure" is meant that SP18 monomer is present as part ofa composition free of other alveolar surfactant proteins and whereinless than 20 percent, preferably less than 10 percent and morepreferably less than 5 percent, of the SP18 monomer present is inhomodimeric form, i.e., present as part of SP18 dimer.

Preferably, a SP18 monomer-containing composition of the presentinvention contains human SP18 monomer. More preferably, a SP18monomer-containing composition contains SP18 monomer having an aminoacid residue sequence shown in FIG. 1 from about residue position 1 toat least about residue position 75, preferably to at least aboutposition 81. More preferably, a SP18 monomer used to form a subjectprotein composition corresponds in sequence to the sequence shown inFIG. 1 from residue position 1 to residue position 81.

Preferably, the amino acid residue sequence of a SP18 monomer in asubject SP18 monomer-containing composition corresponds to the sequenceof a native SP18 monomer. However, it should be understood that a SP18monomer used to form a protein composition of the present invention neednot be identical to the amino acid residue sequence of a native SP18monomer, but may be subject to various changes, such as those describedhereinbelow for a polypeptide of this invention, so long as suchmodifications do not destroy surfactant activity. Such modified proteincan be produced, as is well known in the art, through, for example,genomic site-directed mutagenesis.

"Surfactant activity" for a protein or polypeptide is defined as theability, when combined with lipids, either alone or in combination withother proteins, to exhibit activity in the in vivo assay of Robertson,Lung, 158:57-68 (1980). In this assay, the sample to be assessed isadministered through an endotracheal tube to fetal rabbits or lambsdelivered prematurely by Caesarian section. (These "preemies" lack theirown PS, and are supported on a ventilator.) Measurements of lungcompliance, blood gases and ventilator pressure provide indices ofactivity. Preliminary assessment of activity may also be made by an invitro assay, for example that of King, et al., Am. J. Physiol.,223:715-726 (1972), or that illustrated below which utilizes ameasurement of surface tension at a air-water interface when a proteinor polypeptide is admixed with a phospholipid.

C. Nucleic Acid Segments

In living organisms, the amino acid residue sequence of a protein orpolypeptide is directly related via the genetic code to thedeoxyribonucleic acid (DNA) sequence of the structural gene that codesfor the protein. Thus, a structural gene can be defined in terms of theamino acid residue sequence, i.e., protein or polypeptide, for which itcodes.

An important and well known feature of the genetic code is itsredundancy. That is, for most of the amino acids used to make proteins,more than one coding nucleotide triplet (codon) can code for ordesignate a particular amino acid residue. Therefore, a number ofdifferent nucleotide sequences may code for a particular amino acidresidue sequence. Such nucleotide sequences are considered functionallyequivalent since they can result in the production of the same aminoacid residue sequence in all organisms. Occasionally, a methylatedvariant of a purine or pyrimidine may be incorporated into a givennucleotide sequence. However, such methylations do not affect the codingrelationship in any way.

A DNA segment of the present invention is characterized as consistingessentially of a DNA sequence that encodes a SP18 monomer, preferablyhuman SP18 monomer. That is, a DNA segment of the present inventionforms a structural gene capable of expressing a SP18 monomer. While thecodons of the DNA segment need not be collinear with the amino acidresidue sequence of SP18 monomer because of the presence of an intron,it is preferred that the structural gene is capable of expressing SP18monomer in mature form, i.e., without the need for post-translationalproteolytic processing. Preferably, the gene is present as anuninterrupted linear series of codons where each codon codes for anamino acid residue found in a SP18 monomer, i.e., a gene containing nointrons.

Thus, a DNA segment consisting essentially of the sequence shown in FIG.1 from about nucleotide position 187 to about nucelotide position 426,preferably to about nucelotide position 429, and capable of expressingSP18 monomer, constitutes one preferred embodiment of the presentinvention.

DNA segments that encode SP18 monomer can easily be synthesized bychemical techniques, for example, the phosphotriester method ofMatteucci, et al., J. Am. Chem. Soc., 103:8185 (1981). Of course, bychemically synthesizing the coding sequence, any desired modificationscan be made simply by substituting the appropriate bases for theseencoding the native amino acid residue sequence.

Also contemplated by the present invention are ribonucleic acid (RNA)equivalents of the above described DNA segments.

D. Recombinant Nucleic Acid Molecules

The recombinant nucleic acid molecules of the present invention can beproduced by operatively linking a vector to a nucleic acid segment ofthe present invention.

As used herein, the phase "operatively linked" means that the subjectnucleic acid segment is attached to the vector so that expression of thestructural gene formed by the segment is under the control of thevector.

As used herein, the term "vector" refers to a nucleic acid molecularcapable of replication in a cell and to which another nucleic acidsegment can be operatively linked so as to bring about replication ofthe attached segment. Vectors capable of directing the expression of astructural gene coding for SP18 monomer are referred to herein as"expression vectors." Thus, a recombinant nucleic acid molecule (rDNA orrRNA) is a hybrid molecular comprising at least two nucleotide sequencesnot normally found together in nature.

The choice of vector to which a nucleic acid segment of the presentinvention is operatively linked depends directly, as is well known inthe art, on the functional properties desired, e.g., protein expression,and the host cell to be transformed, these being limitations inherent inthe art of constructing recombinant nucleic acid moleculars. However, avector contemplated by the present invention is at least capable ofdirecting the replication, and preferably also expression, of SP18monomer structural gene included in a nucleic acid segment to which itis operatively linked.

In preferred embodiments, a vector contemplated by the present inventionincludes a procaryotic replicon, i.e., a DNA sequence having the abilityto direct autonomous replication and maintenance of an rDNA moleculeextrachromasomally in a procaryotic host cell, such as a bacterial hostcell, transformed therewith. Such replicons are well known in the art.In addition, those embodiments that include a procaryotic replicon alsoinclude a gene whose expression confers drug resistance to a bacterialhost transformed therewith. Typical bacterial drug resistance genes arethose that confer resistance to ampicillin or tetracycline.

Those vectors that include a procaryotic replicon can also include aprocaryotic promoter capable of directing the expression (transcriptionand translation) of a SP18 monomer gene in a bacterial host cell, suchas E. coli, transformed therewith. A promoter is an expression controlelement formed by a DNA sequence that permits binding of RNA polymeraseand transcription to occur. Promoter sequences compatible with bacterialhosts are typically provided in plasmid vectors containing convenientrestriction sites for insertion of a DNA segment of the presentinvention. Typical of such vector plasmids are pUC8, pUC9, pBR322 andpBR329 available from Biorad Laboratories, (Richmond, Calif.) and pPLand pKK223 available from Pharmacia, Piscataway, N.J.

Expression vectors compatible with eucaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form an rDNAmolecule of the present invention. Eucaryotic cell expression vectorsare well known in the art and are available from several commercialsources. Typically, such vectors are provided containing convenientrestriction sites for insertion of the desired DNA segment. Typical ofsuch vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d(International Biotechnologies, Inc.), and pTDT1 (ATCC, #31255).

In preferred embodiments, a eucaryotic cell expression vector used toconstruct an rDNA molecule of the present invention includes a selectionmarker that is effective in a eucaryotic cell, preferably a drugresistance selection marker. A preferred drug resistance marker is thegene whose expression results in neomycin resistance, i.e., the neomycinphosphotransferase (neo) gene. Southern, et al., J. Mol. Appl. Genet.,1:327-341 (1982).

The use of retroviral expression vectors to form a recombinant nucleicacid molecule of the present invention is also contemplated. As usedherein, the term "retroviral expression vector" refers to a nucleic acidmolecule that includes a promoter sequence derived from the longterminal repeat (LTR) region of a retrovirus genome.

In preferred embodiments, the expression vector is a retroviralexpression vector that is replication-incompetent in eucaryotic cells.The construction and use of retroviral vectors has been described bySorge, et al., Mol. Cell. Biol., 4:1730-37 (1984).

A variety of methods have been developed to operatively link nucleicacid segments to vectors via complementary cohesive termini. Forinstance, complementary homopolymer tracts can be added to the nucleicacid segment to be inserted and to a terminal portion of the vectornucleic acid. The vector and nucleic acid segment are than joined byhydrogen bonding between the complementary homopolymeric tails to form arecombinant nucleic acid molecule.

Synthetic linkers containing one or more restriction sites provide analternative method of joining a nucleic acid segment to vectors. Forinstance, a DNA segment of the present invention is treated withbacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymesthat remove protruding, 3', single-stranded termini with their 3'-5'exonucleolytic activities and fill in recessed 3' ends with theirpolymerizing activities. The combination of these activities thereforegenerates blunt-ended DNA segments. The blunt-ended segments are thenincubated with a large molar excess of linker molecules in the presenceof an enzyme that is able to catalyze the ligation of blunt-ended DNAmolecules, such as bacterophage T4 DNA ligase. Thus, the products of thereaction are DNA segments carrying polymeric linker sequences at theirends. These DNA segments are then cleaved with the appropriaterestriction enzyme and ligated to an expression vector that has beencleaved with an enzyme that produces termini compatible with those ofthe DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies, Inc., New Haven, Conn.

Also contemplated by the present invention are RNA equivalents of theabove described recombinant DNA molecules.

E. Transformed Cells and Cultures

The present invention also relates to a host cell transformed with arecombinant nucleic acid molecule of the present invention, preferablyan rDNA capable of expressing an SP18 monomer. The host cell can beeither procaryotic or eucaryotic.

"Cells" or "transformed host cells" or "host cells" are often usedinterchangeably as will be clear from the context. These terms includethe immediate subject cell, and, of course, the progeny thereof. It isunderstood that not all progeny are exactly identical to the parentalcell, due to chance mutations or differences in environment. However,such altered progeny are included when the above terms are used.

Bacterial cells are preferred procaryotic host cells and typically are astrain of E. coli such as, for example, the E. coli strain DH5 availablefrom Bethesda Research Laboratories, Inc., Bethesda, Md. Preferredeucaryotic host cells include yeast and mammalian cells, preferablyvertebrate cells such as those from a mouse, rat, monkey or humanfibroblastic cell line. Preferred eucaryotic host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61 and NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658.Transformation of appropriate cell hosts with a recombinant nucleic acidmolecule of the present invention is accomplished by well known methodsthat typically depend on the type of vector used. With regard totransformation of procaryolytic host cells, see, for example, Cohen, etal., Proc. Natl. Acad. Sci. USA, 69:2110 (1972); and Maniatis, et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982). With regard to transformation ofvertebrate cells with recombinant nucleic acid molecules containingretroviral vectors, see, for example, Sorge, et al., Mol. Cell. Biol.,4:1730-37 (1984); Graham, et al., Virol., 52:456 (1973); and Wigler, etal., Proc. Natl. Acad. Sci. USA, 76:1373-76 (1979).

Successfully transformed cells, i.e., cells that contain a recombinantnucleic acid molecule of the present invention, can be identified bywell known techniques. For example, cells resulting from theintroduction of an rDNA of the present invention can be cloned toproduce monoclonal colonies. Cells from those colonies can be harvested,lysed and their DNA content examined for the presence of the rDNA usinga method such as that described by Southern, J. Mol. Biol., 98:503(1975) or Berent, et al., Biotech., 3:208 (1985).

In addition to directly assaying for the presence of DNA, successfultransformation can be confirmed by well known immunological methods whenthe rDNA is capable of directing the expression of an SP18 monomer. Forexample, cells successfully transformed with an expression vectoroperatively linked to a DNA segment of the present invention produceproteins displaying SP18 monomer antigenicity. Thus, a sample of a cellculture suspected of containing transformed cells are harvested andassayed for human SP18 using antibodies specific for that antigen, theproduction and use of such antibodies being well known in the art.

Thus, in addition to the transformed host cells themselves, the presentinvention also contemplates a culture of those cells, preferably amonoclonal (clonally homgenous) culture, or a culture derived from amonoclonal culture, in a nutrient medium. Preferably, the culture alsocontains a protein displaying SP18 monomer antigenicity, and morepreferably, biologically active SP18 monomer.

Nutrient media useful for culturing transformed host cells are wellknown in the art and can be obtained from several commercial sources. Inembodiments wherein the host cell is mammalian, a "serum-free" medium ispreferably used.

F. Recombinant Methods for Producing SP18

Another aspect of the present invention pertains to a method forproducing SP18, preferably human SP18 monomer. The method entailsinitiating a culture comprising a nutrient medium containing host cells,preferably human cells, transformed with a rDNA molecule of the presentinvention that is capable of expressing SP18 monomer. The culture ismaintained for a time period sufficient for the transformed cells toexpress SP18 monomer. The expressed protein is then recovered from theculture.

Methods for recovering an expressed protein from a culture are wellknown in the art and include fractionation of the protein-containingportion of the culture using well known biochemical techniques. Forinstance, the method of gel filtration, gel chromatography,ultrafiltration, electrophoresis, ion exchange, affinity chromatographyand the like, such as are known for protein fractionations, can be usedto isolate the expressed proteins found in the culture. In addition,immunochemical methods, such as immunoaffinity, immunoadsporption andthe like can be performed using well known methods.

Also contemplated by the present invention is an SP18 monomer producedby a recombinant nucleic acid method described herein.

G. Polypeptides

A polypeptide of the present invention (subject polypeptide) ischaracterized by its amino acid residue sequence and novel functionalproperties. A subject polypeptide when admixed with a pharmaceuticallyacceptable phospholipid forms a synthetic pulmonary surfactant having asurfactant activity greater than the surfactant activity of thephospholipid alone (as indicated by a lower ΔP as shown in FIG. 6 and ahigher volume per given pressure as shown in FIGS. 7 and 8).

As seen in FIG. 1, SP18 has a large hydrophobic region (residues 1 toabout 75), followed by a relatively short hydrophilic region at thecarboxy terminus (residues 76 to 80, or 81). In referring to amino acidresidue numbers of the SP18 sequence, those residues are as illustratedin FIG. 1.

In one embodiment, a subject polypeptide consists essentially of atleast about 10, preferably at least 11 amino acid residues, and no morethan about 60, more usually few than about 35 and preferably fewer thanabout 25 amino acid residues that correspond to the sequence of SP18monomer.

Usually, the amino acid sequence of a polypeptide of this invention willcorrespond to a single group of contiguous residues in the linearsequence of SP18. However, polypeptides that correspond to more than oneportion of the SP18 sequence are also contemplated. Usually at least onesequence that corresponds to at least 10, preferably at least 15,contiguous residues of the hydrophobic region of SP18 will be present inthe peptide. A plurality of hydrophobic region amino acid sequences maybe present.

A subject polypeptide will preferably include as its carboxy terminalsequence at least 5 contiguous residues in the linear sequence of SP18including residue 80. Thus the polypeptides of this invention mayinclude one or more groups of amino acid residues that correspond toportion of SP18 so that a sequence corresponding to a first group ofcontiguous residues of the SP18 monomer may be adjacent to a sequencecorresponding to a second group of contiguous residues from the same oranother portion of the SP18 monomer in the polypeptide sequence.Peptides having two or more sequences that correspond to a single groupof contiguous amino acid residues from the linear sequence of SP18 isalso contemplated.

Exemplary preferred subject polypeptides corresponding in amino acidresidue sequence to human SP18 monomer hydrophobic region are shown inTable 1.

                  TABLE 1                                                         ______________________________________                                        Desig-                                                                        nation.sup.1                                                                        Amino Acid Residue Sequence                                             ______________________________________                                        p1-15 FPIPLPYCWLCRALI                                                         p11-25                                                                              CRALIKRIQAMIPKG                                                         p21-35                                                                              MIPKGALAVAVAQVC                                                         p31-45                                                                              VAQVCRVVPLVAGGI                                                         p41-55                                                                              VAGGICQCLAERYSV                                                         p46-76                                                                                 CQCLAERYSVILLDTLLGRMLPQLVCRLVLR                                      p51-65                                                                                    ERYSVILLDTLLGRM                                                   p51-72                                                                                    ERYSVILLDTLLGRMLPQLVCR                                            P51-76                                                                                    ERYSVILLDTLLGRMLPQLVCRLVLR                                        p54-72                                                                                      SVILLDTLLGRMLPQLVCR                                             p54-76                                                                                      SVILLDTLLGRMLPQLVCRLVLR                                         p61-75                                                                                          LLGRMLPQLVCRLVL                                             ______________________________________                                         .sup.1 The designation of each peptide inidicates that portion of the         amino acid residue sequence of human SP18 monomer, as shown in FIG. 1 to      which the peptide sequence corresponds, i.e., it indicates the location o     the peptide sequence in the protein sequence.                            

In preferred embodiments, a subject polypeptide is further characterizedas having a carboxy-terminal amino acid residue sequence represented bythe formula:

    --RLVLRCSMDD.sub.Z,

wherein Z is an integer having a value of 0 or 1 such that when Z is 0the D residue to which it is a subscript is absent and when Z is 1 the Dresidue to which it is a subscript is present. Exemplary preferred"carboxy-terminal polypeptides" are shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Designation                                                                         Amino Acid Residue Sequence                                             __________________________________________________________________________    p71-81                                                                              CRLVLRCSMDD                                                             p66-81                                                                              LPQLVCRLVLRCSMDD                                                        p59-81                                                                              DTLLGRMLPQLVCRLVLRCSMDD                                                 p52-81                                                                              RYSVILLDTLLGRMLPQLVCRLVLRCSMDD                                          P51-81                                                                              ERYSVILLDTLLGRMLPQLVCRLVLRCSMDD                                         P51-80                                                                              ERYSVILLDTLLGRMLPQLVCRLVLRCSMD                                          p36-81                                                                              RVVPLVAGGICQCLAERYSVILLDTLLGRMLPQLVCRLVLRCSMDD                          p32-81                                                                              AQVCRVVPLVAGGICQCLAERYSVILLDTLLGRMLPQLVCRLVLRCSMDD                      __________________________________________________________________________     .sup.1 The designation is the same as in Table 1.                        

Preferably, a subject polypeptide has an amino acid residue sequencethat corresponds to a portion of the sequence shown in FIG. 1. However,it should be understood that a polypeptide of the present invention neednot be identical to the amino acid residue sequence of a native SP18monomer. Therefore, a polypeptide of the present invention can besubject to various changes, such as insertions, deletions andsubstitutions, either conservative or non-conservative, where suchchanges provide for certain advantages in their use.

Conservative substitutions are those where one amino acid residue isreplaced by another, biologically similar residue. Examples ofconservative substitutions include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine or another, orthe substitution of one polar residue for another such as betweenarginine and lysine, between glutamic and aspartic acids or betweenglutamine and asparagine and the like. The term "conservativesubstitution" also includes the use of a substituted amino acid in placeof an unsubstituted parent amino acid provided that such a polypeptidealso displays the requisite binding activity.

In one preferred embodiment, a serine (S) residue is substituted for acysteine (C) residue, usually at least one of residue positions 71 and77. Preferably the serine analog has a sequence corresponding to thesequence of residues 51-76 of the SP18 monomer with the substitution atresidue 71 or to the sequence of residues 51-81 with serinesubstitutions at 71 and 77.

When a polypeptide of the present invention has a sequence that is notidentical to the sequence of a native SP18 monomer because one or moreconservative or non-conservative substitutions have been made, usuallyno more than about 20 number percent and more usually no more than 10number percent of the amino acid residues are substituted, except whereadditional residues have been added at either terminus as for thepurpose of providing a "linker" by which the polypeptides of thisinvention can be conveniently affixed to a label or solid matrix, orcarrier. Labels, solid matrices and carriers that can be used with thepolypeptides of this invention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues that do not correspondin amino acid residue sequence to a native SP18 monomer. Typical aminoacid residues used for linking are tyrosine, cysteine, lysine, glutamicand aspartic acid, or the like. In addition, a polypeptide sequence ofthis invention can differ from the natural sequence by the sequencebeing modified by terminal-NH₂ acylation, e.g., acetylation, orthioglycolic acid amidation, terminal-carboxylyamidation, e.g., ammonia,methylamine, etc.

When coupled to a carrier via a linker to form what is known in the artas a carrier-hapten conjugate, a polypeptide of the present invention iscapable of inducing antibodies that immunoreact with SP18 monomer. Inview of the well established principle of immunologic cross-reactivity,the present invention therefore contemplates antigenically relatedvariants of the polypeptides shown in Tables 1 and 2. An "antigenicallyrelated variant" is a polypeptide that includes at least a six aminoacid residue sequence portion of a polypeptide from Table 1 or Table 2and which is capable of inducing antibody molecules that immunoreactwith a polypeptide from Table 1 or 2 and an SP18 monomer.

In another embodiment, a polypeptide of this invention has amino acidresidue sequence that has a composite hydrophobicity less than zero,preferably less than or equal to -1, more preferably less than or equalto -2. Determination of the composite hydrophobicity value for a peptideis described in detail in Example 2. These hydrophobic polypeptidesperform the function of the hydrophobic region of SP18. In a preferredembodiment, the amino acid sequence mimics the pattern of hydrophobicand hydrophilic residues of SP18.

A preferred hydrophobic polypeptide includes a sequence havingalternating hydrophobic and hydrophilic amino acid residue regions andis characterized as having at least 10 amino acid residues representedby the formula:

    (Z.sub.a U.sub.b).sub.c Z.sub.d

Z and U are amino acid residues such that at each occurrence Z and U areindependently selected. Z is a hydrophilic amino acid residue,preferably selected from the group consisting of R, D, E and K. U is ahydrophilic amino acid residue, preferably selected from the groupconsisting of V, I, L, C, Y and F. "a", "b", "c" and "d" are numberswhich indicate the number of hydrophilic or hydrophobic residues. "a"has an average value of about 1 to about 5, preferably about 1 to about3. "b" has an average value of about 3 to about 20, preferably about 3to about 12, most preferably 2 to 10, most preferably 3 to 6. "d" is 1to 3, preferably 1 to 2.

By stating that the amino acid residue represented by Z and U isindependently selected, it is meant that at each occurrence a residuefrom the specified group is selected. That is, when "a" is 2, forexample, each of the hydrophilic residues represented by Z will beindependently selected and thus can include RR, RD, RE, RK, DR, DD, DE,DK, etc. By stating that "a" and "b" have average values, it is meantthat although the number of residues within the repeating sequence,(Z_(a) U_(b)) can vary somewhat within the peptide sequence, the averagevalues of "a" and "b" would be about 1 to about 5 and about 3 to about20, respectively.

Exemplary preferred polypeptides of the above formula are shown in Table3.

                  TABLE 3                                                         ______________________________________                                        Designation.sup.1                                                                        Amino Acid Residue Sequence                                        ______________________________________                                        DL4        DLLLLDLLLLDLLLLDLLLLD                                              RL4        RLLLLRLLLLRLLLLRLLLLR                                              RL8        RLLLLLLLLRLLLLLLLLRLL                                              RL7        RRLLLLLLLRRLLLLLLLRRL                                              RCL1       RLLLLCLLLRLLLLCLLLR                                                RCL2       RLLLLCLLLRLLLLCLLLRLL                                              RCL3       RLLLLCLLLRLLLLCLLLRLLLLCLLLR                                       ______________________________________                                         .sup.1 The designation is an abbreviation for the indicated amino acid        residue sequence.                                                        

Also contemplated are composite polypeptides of 10 to 60 amino acidresidues. A composite polypeptide consists essentially of an aminoterminal sequence and a carboxy terminal sequence. The amino terminalsequence has an amino acid sequence of a hydrophobic region polypeptideor a hydrophobic peptide of this invention, preferably hydrophobicpeptide, as defined in the above formula. The carboxy terminal sequencehas the amino acid residue sequence of a subject carboxy terminalpeptide.

A polypeptide of the present invention can be synthesized by anytechniques that are known to those skilled in the polypeptide art. Anexcellent summary of the many techniques available may be found in J. M.Steward and J. D. Young, "Solid Phase Peptide Synthesis", W. H. FreemanCo., San Francisco, 1969, and J. Meienhofer, "Hormonal Proteins andPeptides", Vol. 2, p. 46, Academic Press (New York), 1983 for solidphase peptide synthesis, and E. Schroder and K. Kubke, "The Peptides",Vol., 1, Academic Press (New York), 1965 for classical solutionsynthesis.

In general, these method comprise the sequential addition of one or moreamino acid residues or suitably protected amino acid residues to agrowing peptide chain. Normally, either the amino or carboxyl group ofthe first amino acid residue is protected by a suitable, selectivelyremovable protecting group. A different, selectively removableprotecting group is utilized for amino acids containing a reactive sidegroup such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the finalpolypeptide.

H. Synthetic Surfactants

Recombinantly produced SP18 and/or a subject polypeptide can be admixedwith a pharmaceutically acceptable phospholipid to form a syntheticpulmonary surfactant (PS) useful in the treatment of respiratorydistress syndrome.

The phase "pharmaceutically acceptable" refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human.

Phospholipids useful in forming synthetic alveolar surfactants byadmixture with protein are well known in the art. See, Notter, et al.,Clin. Perinatology, 14:433-79 (1987), for a review of the use of bothnative and synthetic phospholipids for synthetic surfactants.

In one embodiment, the present invention contemplates a syntheticpulmonary surfactant effective in treating RDS comprising an effectiveamount of a subject polypeptide admixed with a pharmaceuticallyacceptable phospholipid. While methods for determining the optimalpolypeptide:phospholipid weight ratios for a givenpolypeptide:phospholipid combination are well known, therapeuticallyeffective ratios are in the range of about 1:5 to about 1:10,000,preferably about 1:100 to about 1:5,000, and more preferably about 1:500to about 1:1000. In a more preferred embodiment, thepolypeptide:phospholipid weight ratio is in the range of about 1:5 toabout 1:2,000, preferably about 1:7 to about 1:1,000, and morepreferably about 1:10 to about 1:100. Thus, a synthetic pulmonarysurfactant of this invention can contain about 50, usually about 80, toalmost 100 weight percent lipid and about 50, usually about 20, to lessthan 1 weight percent polypeptide. Preferably a subject polypeptide isabout 1 to about 10 weight percent of the surfactant for polypeptidescorresponding to portions of the SP18 sequence and 1:100 forpolypeptides corresponding to the entire SP18 monomer.

The lipid portion is preferably about 50 to about 90, more preferablyabout 50 to about 75, weight percent dipalmitoylphosphatidylcholine(DPPC) with the remainder unsaturated phosphatidyl choline, phosphatidylglycerol (PG), triacylglycerols, palmitic acid sphingomyelin oradmixtures thereof. The synthetic pulmonary surfactant is prepared byadmixing a solution of a subject polypeptide with a suspension ofliposomes or by admixing the subject polypeptide and lipids directly inthe presence of organic solvent. The solvent is then removed by dialysisor evaporation under nitrogen and/or exposure to vacuum.

A subject synthetic pulmonary surfactant is preferably formulated forendotracheal administration, e.g., typically as a liquid suspension, asa dry powder "dust", or as in aerosol. For instance, a syntheticsurfactant (polypeptide-lipid micelle) is suspended in a liquid with apharmaceutically acceptable excipient such as water, saline, dextrose,glycerol and the like. A surfactant-containing therapeutic compositioncan also contain small amounts of non-toxic auxiliary substances such aspH buffering agents including sodium acetate, sodium phosphate and thelike. To prepare a synthetic surfactant in dust form, a syntheticsurfactant is prepared as described herein, then lyophilized andrecovered as a dry powder.

If it is to be used in aerosol administration, a subject syntheticsurfactant is supplied in finely divided form along with an additionalsurfactant and propellant. Typical surfactants which may be administeredare fatty acids and esters. However, it is preferred, in the presentcase, to utilize the other components of the surfactant complex DPPC andPG. Useful propellants are typically gases at ambient conditions, andare condensed under pressure. Lower alkane and fluorinated alkane, suchas Freon, may be used. The aerosol is packaged in a container equippedwith a suitable valve so that the ingredients may be maintained underpressure until released.

A synthetic surfactant is administered, as appropriate to the dosageform, by endotracheal tube, by aerosol administration, or bynebulization of the suspension or dust into the inspired gas. Amounts ofsynthetic PS between about 1.0 and about 400 mg/kg, preferably about 50mg to about 500 mg/kg, are administered in one dose. For use in newlyborn infants, one to three administrations are generally sufficient. Foradults, sufficient reconstituted complex is administered to produce aPO₂ within the normal range (Hallman, et al., J. Clinical Investigation,70:673-682, 1982).

The following examples are intended to illustrate, but not limit, thepresent invention.

EXAMPLES Example 1--Isolation and Characterization of Native SP18 A.Methods

Purification of LMW apoproteins

Human pulmonary surfactant was isolated from full-term amniotic fluidand applied to a column of DEAE-Sephacel A-50 (Pharmacia, Uppsala,Sweden) using 4 milliliter (ml) packed volume per 200 milligram (mg)surfactant, in a tris-EDTA buffer containing 1%n-octyl-beta-D-glucopyranoside as described by Revak, et al., Am. Rev.Respir. Dis., 134:1258-1265 (1986) and Hallman, et al., Pediatrics,71:473-482 (1983). This particular column and conditions were used inorder to isolate the 35,000 dalton apoprotein (for use in other studies)without exposing it to potentially denaturing organic solvents. The voidvolume, containing the lipids and proteins which did not bind to thecolumn under these conditions, was pooled and extracted with an equalvolume of 2:1 chloroform:methanol.

Following centrifugation to separate the phases, the upper phase(water+methanol) was re-extracted with 1/2 volume chloroform. Aftercentrifugation, the resultant lower organic phase was added to theinitial lower phase and evaporated to dryness under a stream ofnitrogen. This extract, which contained 100-180 mg phospholipid, LMWapoproteins, and octylglucopyranoside, was redissolved in 2.5 ml ofchloroform:methanol, 2:1.

Following the method of Takahashi, et al., Biochem. Biophys. Res. Comm.,135:527-532 (1986), which was found to afford a good separation ofoctylglucopyranoside from the LMW proteins and phospholipids, a glasscolumn 2.5 cm in diameter was packed at 4° to a height of 38 cm withSephadex LH-20 (Pharmacia, Uppsala, Sweden) in 2:1 chloroform:methanol.The sample was loaded and 2 ml fractions collected aschloroform:methanol, 2:1, was run through at a flow rate of 8.5 ml/hr.Phospholopids eluted after 40 ml of buffer had passed through thecolumn. Octylglycopyranoside appeared at the 56-116 ml region.

The phospholopid region was pooled, dried under nitrogen, andredissolved in 1 ml chloroform. A silicic acid column was prepared bypacking 9 ml of Bio-Sil HA (BioRad, Richmond, Calif.) in chloroform in aglass column at room temperature. The sample (which containedapproximately 50 mg phospholopid) was applied and washed with 11 mlchloroform. A linear gradient of increasing methanol was establishedusing an equal weight of chloroform and methanol (38.8 g, 26.5 mlchloroform and 50 ml methanol). Fractions of 2 ml were collected as thegradient was applied to the column. FIG. 1 shows the protein andphospholipid profiles obtained.

Phospholopid analyses showed a small peak in fractions 17-20 and a majorpeak after fraction 30. The Pierce BCA protein assay was positive infractions 12-19 and 28-33, but it should be noted that the latter peakis likely to be due to the phospholipid present in this region.Electrophoresis in sodium dodecyl sulfate polyacrylamide gels showed theLMW apoproteins were present in fractions 13-19 with some separationoccurring between SP9 and SP18.

Alternatively, a method devised by Hawgood, et al., Proc. Natl. Acad.Sci., 85:66-70 (1987) employing a butanol extraction of PS followed bychromatography of Sephadex LH-20 in an acidified chloroform/methanolbuffer, can be used to isolate the LMW apoprotein mixture. For somestudies, a separation of the two LMW apoproteins was effected usingSephadex LH-60. A glass column of 1 cm diameter was packed to 40 cm withSephadex LH-60 (Pharmacia, Uppsala, Sweden) in chloroform/methanol, 1:1,containing 5% 0.1 N HCl. A flow rate of 1-2 ml/hr was used. A mixture ofthe LMW apoproteins containing about 200-700 micrograms (ug) of proteinfrom either the Bio-Sil HA column or the LH-20 column described byHawgood, et al., Proc. Natl. Acad. Sci., 85:66-70 (1987), in a volume of0.5 ml buffer, was applied to the top of the column and fractions of 0.5ml were collected. Typically, SP18 protein eluted in fractions 16-19 andSP9 in fractions 24-29. Appropriate fractions were pooled and dried inglass tubes under nitrogen. A brief period of lyophilization ensuredcomplete removal of the HCl. Proteins were re-solubilized in methanolprior to use.

SDS-Gel Electrophoresis

Gel electrophoresis in 16% polyacrylamide was performed in the presenceof sodium dodecyl sulfate (SDS-PAGE) according to the method of Laemmli,Nature, 227:680-685 (1970), using 3×7 cm minislab gels. 1%β-mercaptoethanol was added to samples where indicated as a disulfidereducing agent. Following electrophoresis, the gels were fixed overnightin 50% methanol+12% acetic acid, washed in water for 2 hours, andsilver-stained according to the method of Wray, et al., Anal. Biochem.,118:197-203 (1981).

Octylglucopyranoside Assay

An assay for the quantitation of n-octyl-beta-D-glyco-pyranoside, basedon the anthrone method of Spiro, Methods Enzymol., 8:3-5 (1966) has beendescribed previously by Revak, et al., Am. Rev. Respir. Dis.,134:1258-1265 (1986).

Protein Determinations

Organic samples containing up to 5 ug protein were dried in 12×75 mmglass tubes under nitrogen. Fifteen microliters (ul) of 1% SDS in H₂ Oand 300 ul BCA Protein Assay Reagent (Pierce Chemical Co., Rockford,Ill.) were admixed with the protein in each tube. Tubes were covered andincubated at 60° C. for 30 min. After cooling, the samples weretransferred to a 96-well flat-bottom polystyrene microtiter plate andOD₅₅₀ measured. Bovine serum albumin was used as a standard. It shouldbe noted that some phospholipids will react in the BCA protein assaymaking protein quantitations inaccurate when lipid is present (i.e.,prior to Bio-Sil HA chromatography). Additionally, once purified, thehydrophobic LMW apoproteins themselves react poorly with the BCAreagents and all quantitations of the isolated proteins were, therefore,based on amino acid compositions.

Phospholipids

Dipalmitoylphosphatidylcholine (DPPC, beta,gamma-dipalmitoyl-L-alpha-lecithin) and L-alpha-phosphatidyl-DL-glycerol(PG, derivative of egg lecithin) were purchased from eitherCalbiochem-Behring (La Jolla, Calif.) or Avanti Polar-Lipids, Inc.(Birmingham, Ala.). DPPC was added to PG in chloroform in a weight ratioof 3:1.

Admixture of LMW Apoproteins with Phospholipids

For in vitro assays, a methanol solution containing 4 ug of SP9 or SP18,was added to 400 ug DPPC:PG in chloroform in a 12×75 mm glass tube.Following a brief vortex mixing, the samples were dried under N₂. Ninetymicroliters of water were added to each and the tubes placed in a 37° C.water bath for 15 minutes, with periodic gentle mixing. Isotonicity wasrestored with the addition of 10 ul of 9% NaCl to each sample prior toassay. For in vivo rabbit studies, 50 ug LMW apoproteins (containingboth SF9 and SP18) or 25 ug SP9 or 25 ug SP18 were dried under N₂. Fivemg of phospholipids (DPPC:PG, 3:1) were added in chloroform. The sampleswere mixed, dried, and resuspended in 250 ul 100 millimolar (mM) salinecontaining 1.5 mM CaCl₂, to yield reconstituted surfactant at 20 mg/mlwith 0.5-1% protein.

Surfactant Activity Assays

In vitro assays of surfactant activity, assessed as its ability to lowerthe surface tension of a pulsating bubble, and in vivo assays utilizingfetal rabbits, have both been described in detail previously by Revak,et al., Am. Rev. Respir. Dis., 134:1258-1265 (1986).

Morphometric Analyses

Fetal rabbit lungs, inflated to 30 cm H₂ O and then deflated to 10 cm H₂O, were submerged in 10% formalin for 72 hours. Paraffin sections wereoriented from apex to base and 5 micron sections taken anterior toposterior. After hematoxylin and eosin staining, 10 fields (100×) werepoint-counted from apex to base on multiple sections. Standardizedmorphometric methods (Weiber, in "Stereological Methods," Vol. I,Academic Press, New York, pp. 33-58, 1979) were used to determine ratiosof lung interstituium to air spaces for each treatment group.Intersections of alveolar permieters were also determined.

Phospholipid Phosphorus Assays

Phospholipids were quantitated according to the method of Bartlett, J.Biol. Chem., 234:466-468 (1959).

Amino Acid Analysis

Triplicate samples for amino acid compositions were hydrolyzed with HClat 110° C. for 24 hours, with HCl at 150° C. for 24 hours, or inperformic acid at 110° C. for 24 hours followed by HCl hydrolysis at110° C. for 24 hours. Analyses were performed on a Beckman model 121-Mamino acid analyzer (Beckman Instruments, Fullerton, Calif.). Tryptophanwas not determined.

Amino Acid Sequencing

Vapor-phase protein sequencing was performed on an Applied Biosystems470A Amino Acid Sequencer (Applied Biosystems, Inc., Foster City,Calif.) with an on-line Model 120A HPLC.

Isolation of cDNA Clones for Human SP18

RNA was prepared according to Chirgwin, et al., Biochemistry,18:5294-5299 (1979) unaffected adult lung tissue obtained duringsurgical removal of a neoplastic lesion. Preparation of double strandedcDNA was carried out using standard techniques (Chirgwin et al., supra,and Efstratiadis et al., in "Genetic Engineering", eds. Stelow andHollaender, Plenum, N.Y., 1:15-49 (1979) and a library was constructedin lambda NM607 as described by Le Bouc, et al., F.E.B.S. Letts.,196:108-112 (1986). SP18 clones were identified by screening phageplaques with synthetic oligonucleotide probes (Benton, et al., Science,196:180-182 (1977) which were prepared using an Applied Biosystemsautomated synthesizer and purified by HPLC. Initial candidate cloneswere obtained using probe TG996 (5'CATTGCCTGTGGTATGGCCTGCCTCC 3') whichwas derived from the partial nucleotide sequence of a small humansurfactant apoprotein cDNA (Schilling et al., International PatentApplication WO 86/03408). Larger (5'TCGAGCAGGATGACGGAGTAGCGCC 3') whichwas based on the 5' sequence of one of the original clones. Thenucleotide sequence of the cDNA clones was determined by the chaintermination method (Sanger, et al., Proc. Natl. Acad. Sci. U.S.A.,74:5463-5467 (1977) using EcoRI restriction fragments subcloned in anappropriate M13 vector.

B. Results

Characteristics of the LMW Apoproteins

The LMW apoproteins isolated from human amniotic fluid appeared aftersilicic acid chromatography, or after the Sephadex LH-20 columnchromatography FIG. 2 described by Hawgood, et al., Proc. Natl. Acad.Sci. 85:66-70 (1987), as two protein bands in SDS-polyacrylamide getelectrophoresis under non-reducing conditions. The upper band, having anapparent molecular weight of 18,000 daltons is a dimer, and thereforedesignated SP18 dimer. With the addition of β-mercaptoethanol, SP18dimer reduced to 9,000 daltons ad was designated SP18 monomer (FIG. 3).The other LMW apoprotein, designated SP9, appears as a diffuse bandbetween 9 and 12,000 daltons in the presence or absence of reducingagents. SP9 was separated from SP18 dimer and SP18 monomer bychromatography on Sephadex LH-60. The resultant purified proteins areshown in FIG. 3.

Amino acid compositions were determined for SP18 monomer and SP9.Because of the extremely hydrophobic nature of these proteins, HClhydrolysis was performed at 150° C. for 24 hours, in addition to thestandard 110° C. for 24 hour hydrolysis, and values for valine, leucine,and isoleucine calculated from analyses of the hydrolysates done underthe extreme conditions. As shown in Table 3, both proteins are extremelyhydrophobic with high levels of valine and leucine.

                  TABLE 3                                                         ______________________________________                                        Amino Acid Composition of Human SP9 an SP18 monomer                           and a Comparison with the Theoretical Composition of                          SP18.sup.1                                                                                SP9         SP18     SP18.sup.1                                   Amino Acid  (mole %)    (mole %) (mole %)                                     ______________________________________                                        Aspartic acid                                                                             1.1         3.4      3.7                                          (or Asparagine)                                                               Threonine   0.8         1.5      1.2                                          Serine      1.8         2.7      2.5                                          Glutamic acid                                                                             1.5         6.7      6.2                                          (or Glutamine)                                                                Proline     8.3         7.8      7.4                                          Glycine     10.6        6.1      4.9                                          Alanine     4.9         10.2     9.9                                          Cysteine.sup.2                                                                            9.1         7.2      8.6                                          Valine.sup.3                                                                              12.2        11.7     11.1                                         Methionine  3.4         3.2      3.7                                          Isoleucine.sup.3                                                                          6.8         6.4      7.5                                          Leucine.sup.3                                                                             22.4        17.4     17.3                                         Tyrosine    0.7         2.2      2.5                                          Phenylalnine                                                                              2.6         1.5      1.2                                          Histidine   5.4         0.0      0.0                                          Lysine      4.7         3.0      2.5                                          Arginine    3.9         9.0      8.6                                          Tryptophan  N.D..sup.4  N.D..sup.4                                                                             1.2                                          ______________________________________                                         .sup.1 The theoretical composition is based on sequence data through          residue 81.                                                                   .sup.2 Cysteine content was determined following performic acid and HCl       hydrolyses.                                                              

Amino-terminal sequence analysis of SP18 monomer yielded the followingsequence:

    N.sub.2 --Phe--Pro--Ile--Pro--Leu--Pro--Try--.

Repeated sequencing of the purified SP9 monomer showed multiplepeptides, all rich in leucine and containing at least six consecutivevalines. NH₂ -terminal analysis showed phenylalanine, glycine, andisoleucine with the relative amounts of each varying from preparation topreparation.

Nucleotide Sequence Analysis of SP18 cDNA

The nucleotide sequence of a SP18 monomer cDNA clone is presented inFIG. 1. The sequence displays 83% homology with the canine SP18 cDNA(Hawgood, et al., Proc. Natl. Acad. Sci., 85:66-70 (1987). A sequencewithin a large open reading frame was identified which matches perfectlywith the amino terminus of SP18 monomer as determined by Edmandegradation of the isolated protein (underlined in FIG. 3). Thissuggests that mature SP18 monomer arises by processing of a largerprecursor molecule. In the mature sequence there is a single potentialN-glycosylation site (Asn 110), no sites for tyrosine sulfation, and noG-X-Y repeats as found in the 35,000 dalton apoprotein (White, et al.,A. Pediatrics Research, 19:501-508; 1985).

The molecular weight of 9000 daltons obtained by SDS-PAGE of reducedSP18 dimer is lower than that predicted for the complete precursorprotein sequence with amino terminusNH2--Phe--Pro--Ile--Pro--Leu--Pro--Tyr (19,772 daltons), implyingfurther processing in the region of amino acids 70-90. In support ofthis, the theoretical amino acid composition (Table I) of a putative 900dalton protein comprising residues 1 to 81 compares well with thedetermined values for purified SP18 monomer. The amino terminal portionof the precursor protein (residues 1 to 81) is alkaline and morehydrophobic than the COOH terminal portion (residues 82 to 181): theKyte-Doolitle index for residues 1 to 81 is 9100 (pI 8.6) and is -3000(pI 5.91) for residues 82 to 181 (Kyte, et al., J. Pediatrics,100:619-622; 1982). The amino terminus (residues 1 to 81) is, as in thecanine sequence (Hawgood, et al., Proc. Natl. Acad. Sci., 85:66-70;1987), composed of three hydrophobic domains: residues 1 to 11, 22 to 49and 53 to 74. These are interspersed with a charged domain (residues 12to 21) and two hydrophilic and charged stretches (residues 47 to 54 and74 to 81).

Reconstitution of Surfactant Activity with LMW Apoproteins

Samples were prepared containing 400 ug/100 ul phopholipids (DPPC:PG,3:1 by weight), phospholipids plus 4 ug SP9, or phospholipids plus 4 ugSP18. Each sample was assayed in the pulsating bubble surfactometer forthe ability to lower surface tension. The results are shown in Table 4as the mean minimal surface tension at 15 sec, 1 minute (min), and 5min. Natural human surfactant, isolated from term amniotic fluid,diluted to 4 mg/ml is shown for comparison. While neither phospholipidsnor LMW apoproteins alone had significant surface-tension loweringcapacities, a mixture of phospholipids with either SP9 or SP18 showedsignificant activity. Recombining the phospholipids with 1% by weight ofSP18 lowered the surface tensions measured to levels comparable to thoseobtained with an equal amount of natural human surfactant (6.3±0.2dynes/cm for phospholipids+SP18 at 15 sec, 2.0±1.2 dynes/cm for naturalsurfactant). On an equal weight basis, SP9 lowered surface tension lesseffectively (16.7±0.8 dynes/cm at 15 sec).

                  TABLE 4                                                         ______________________________________                                        Minimum Surface Tensions in the Pulsating Bubble.sup.1                                   15 sec  1 min       5 min                                          ______________________________________                                        PL.sup.2     42.9 ± 1.4                                                                           41.6 ± 1.6                                                                             34.9 ± 4.9                              PL + SP9.sup.3                                                                             16.7 ± 0.8                                                                           14.1 ± 1.2                                                                             12.2 ± 1.0                              PL + SP18.sup.3                                                                            6.3 ± 0.2                                                                            5.1 ± 1.0                                                                              4.9 ± 0.6                               natural human                                                                              2.0 ± 1.2                                                                            2.4 ± 1.4                                                                              0.4 ± 0.4                               surfactant.sup.4                                                              ______________________________________                                         .sup.1 Pulsation of 20 cycles/min started 10 sec after bubble formation.      All values are in dynes · cm.sup.-1 and are the average of at        least 3 determinations.                                                       .sup.2 Phospholipids DPPC:PG, 3:1, 4 mg/ml                                    .sup.3 1% by weight compare to phospholipids                                  .sup.4 diluted to 4 mg/ml                                                

In vivo assays of exogenous (synthetic surfactant activity wereperformed by instilling into the airways of immature fetal rabbitssaline solutions containing Ca⁺⁺ alone or with the addition ofphospholipids, phospholipids plus LMW apoproteins, or natural humansurfactant. The animals were ventilated for 30 min and then degassed byplacement in a bell jar under vacuum. The lungs were then inflated togiven pressures and the volume of air required for each pressure wasnoted. The volumes required for given pressure during deflation from 30cm H₂ O were likewise determined. The resulting pressure/volume curvesare shown in FIG. 4 for animals which received synthetic surfactant madewith purified SP9 or SP18 (0.5% by weight compared with totalphospholipid concentration) and appropriate control animals. Improvedlung compliance is apparent in those animals treated with natural oreither synthetic surfactant as compared with those receiving saline orphospholipids with the SP18 appearing more effective than SP9 on anequal weight basis. A similar study was performed using a mixture of SP9and SP18. The results were almost identical to the phospholipid plusSP18 curve presented in FIG. 4.

Following compliance measurements, the lungs were inflated to 30 cm H₂O, deflated back to 10 cm H₂ O, clamped, excised and fixed in formalin.Thin sections were stained with hematoxylin and eosin and examinedmicroscopically. As shown in FIG. 5, lungs treated with saline (A) orphospholipids (C) appeared atelectatic while those from animals whichreceived natural (B) or reconstituted (D) surfactant showed normalalveolar expansion. Morphometric analyses of the thin sections showed aninterstitium to air space ratio of 4.70 for saline treatment and 3.29for phospholipids alone as compared with 0.498 for natural surfactantand 0.538 for reconstituted surfactant. These data are shown in Table 5and corroborate the significant (p<0.001; Mann-Whitney U Test) increasein air space seen in FIG. 5. A comparison of alveolar perimeterssimilarly demonstrated a significantly (p<0.003) greater number ofintersections of the alveolar boundaries in saline- orphospholipid-treated fetuses compared to surfactant-treated animals.

                  TABLE 5                                                         ______________________________________                                        Morphometric Analysis of Airspace                                             Following Fetal Rabbit Treatment                                              Tracheal Instillation                                                                          Interstitium/Air Space                                       ______________________________________                                        saline           4.70                                                         phospholipids.sup.1                                                                            3.29                                                         phospholipids.sup.1 +                                                                          0.538                                                        LMW Apoproteins.sup.2                                                         natural human surfactant.sup.3                                                                 0.498                                                        ______________________________________                                         .sup.1 2 mg of 3:1 DPPC:PG per animal                                         .sup.2 20 ug of LMW apoproteins added to phospholipids                        .sup.3 2 mg per animal                                                   

C. Discussion

This study describes two low molecular weight apoproteins isolated fromhuman amniotic fluid surfactant which can be added to knownphospholipids to produce a biologically active pulmonary surfactant.While the proteins in the current study have been designated as SP18dimer, SP18 monomer and SP9, it is apparent from the recent literaturethat multiple nomenclature and an assortment of reported molecularweights for LMW PS apoproteins (ranging from 5-18,000 daltons) exist.The apparent differences physical properties may be explained by avariety of factors including species differences, varying purificationand handling techniques, varying determinations of low molecular weightsbased on standards in SDS-polyacrylamide gels, and potentialinterference by lipids of low molecular weight protein bands in gels.Comparisons of amino acid compositions and sequences and immunologicanalyses using monospecific antibodies will help to sort out the LMWapoproteins.

It is felt that the SP9 protein described herein, giving a diffuse bandon SDS-polyacrylamide gels from 9-12,000 daltons under reducing ornon-reducing conditions, is probably the same protein as that designatedSAP-6 by Whitsett, et al., Pediatric Research, 20:744-749 (1986), SP5-8by Hawgood, et al., Proc. Natl. Acad. Sci., 85:66-70 (1987), PSP-6 byPhelps, et al., Am. Rev. Respir. Dis., 135:1112-1117 (1987), and the 5kDa proteolipid of Takahashi, et al., Biochem. Biophys Res. Comm.,135:527-532 (1986). The extremely hydrophobic nature of this protein isapparent from its amino acid composition (Table 3) and sequence data,showing at least six consecutive valine residues preceded by aleucine-rich region. The presence of three amino-terminal residues(phenylalanine, glycine, and isoleucine) in the preparations of SP9derived herein from amniotic fluid surfactant suggests a collection ofpeptides having an identical sequence but having had one or two residuesremoved from the amino-terminus. Phelps, et al., Am. Rev. Respir. Dis.,135:1112-1117 (1987) have recently reported a similar finding withbovine PSP-6 apoprotein.

SP18 dimer is comprised of two identical 9000 dalton peptides (butdifferent from the 9000 dalton peptide of SP9) that are disulfidelinked. The amino acid composition of SP18 monomer (Table 3) shows ahigh number of hydrophobic residues. When unreduced SDS-PAGE wereoverloaded with SP18 protein, sequentially less intensely staining bandswere seen at 36,000 and 56,000 daltons, suggesting oligomeric forms ofthe protein; upon reduction, only a single 9000 dalton band was seen.

Both SP9 and SP18 dimer apoproteins isolated as described above, couldbe shown to have biophysical activity following recombination withphospholipids. The addition of 1% by weight of SP18 dimer to thephospholipids DPPC:PG resulted in an immediate increase in surfacepressure resulting in surface tensions of less than 10 dynes/cm by 15sec. The addition of 1% SP9 to DPPC:PG was slightly less effective,lowering surface tensions to 16.7, 14.1, and 12.2 dynes/cm at 15 sec, 1and 5 min, respectively. Mixtures of both SP18 dimer and SP9 were alsoeffective but further studies will be required to determine whether thecombined effect is additive or synergistic.

In vivo studies of reconstituted surfactant using the fetal rabbit model(Schneider, et al., J. Pediatrics, 100:619-622; 1982) were performedusing mixtures of SP18 dimer and SP9 as well as each proteinindividually. A marked improvement in lung compliance was seen inanimals treated with natural surfactant or reconstituted surfactantprepared with SP18 dimer apoprotein, as compared with those receivingphospholipids alone or saline (FIG. 4). A moderate improvement was seenwhen SP9 was used. Identical studies using a mixture of SP18 dimer andSP9 to prepare the reconstituted surfactant showed results very similarto those obtained with SP18 dimer alone (solid squares, FIG. 4);however, the exact ratio of SP18 dimer and SP9 in those studies couldnot be accurately ascertained. FIG. 5 shows representative microscopicalveolar fields indicating the lack of atelectasis following surfactantinstillation.

Suzuki, et al., (Eur. J. Respir. Dis., 69:336-345; 1986) have reported areduction in surface tension (measured on the Wilhelmy balance or in apulsating bubble) and a five fold increase in tidal volumes ofprematurely-delivered rabbits at insufflation pressures of 25 cm and H₂O when porcine LMW (<15,000 daltons) surfactant apoproteins are added tomixtures of DPPC:PG) at a weight ratio of 5:80:20 (protein:DPPC:PG).Whether one or multiple proteins are present in this system is unclear.

Previous studies using the 35,000 dalton apoprotein (Revak, et al., Am.Rev. Respir. Dis., 134:1258-1265; 1986) also showed moderate reductionin surface tension, similar to that obtained with SP9 in the studiesdescribed herein. Clearly, further studies must be done using variouscombinations and concentrations of SP18, SP9 and the 35,000 daltonapoprotein, as well as Ca⁺⁺ and perhaps various phospholipids toelucidate the interactions between these various components ofsurfactant and to determine the best conditions for a biologicallyactive exogenous surfactant.

Example 2--In Vitro Assessment of Polypeptide Surfactant Activity A.Methods

Measurement of Surfactant Activity

Measurements of surface pressure across an air-liquid interface(expressed in negative cm of H₂ O pressure) at minimal bubble radiuswere determined at various times using the pulsating bubble techniquedescribed by Enhorning, J. Appl. Physiol., 43:198-203 (1977).

Briefly, the Enhorning Surfactometer (Surfactometer International,Toronto, Ontario) measures the pressure gradient (ΔP) across aliquid-air interface of a bubble that pulsates at a rate of 20cycles/min between a maximal (0.55 mm) and minimal (0.4 mm) radius. Thebubble, formed in a 37° C., water-enclosed, 20-ul sample chamber, ismonitored through a microscopic optic while the pressure changes arerecorded on a strip chart recorder calibrated for 0 and -2 cm H₂ O.

Determination of Composite Hydrophobicity Value

The composite hydrophobicity value of each peptide was determined byassigning to each amino acid residue in a peptide its correspondinghydrophobicity value as described in Table 1 of Hopp, et al., Proc.Natl. Acad. Sci., U.S.A., 78:3824-3829 (1981), which disclosure isincorporated herein by reference. For a give peptide, the hydrophobicityvalues were summed, the sum representing the composite hydrophobicityvalue.

Preparation of Synthetic Surfactants

After admixture with solvent, each peptide was combined withphospholipids (DPPC:PG), 3:1 to form a synthetic surfactant according toone of the following methods.

Method A was accomplished by admixing 16 ul of peptide/solvent admixture(40 ug peptide) with 100 ul of chloroform containing 400 ugphospholipids, agitating the admixture for about 10 min. at 37° C. toform a peptide/phospholipid admixture. Chloroform was removed from thepeptide/phospholipid admixture by drying under N₂. The syntheticsurfactant thus formed was then admixed with 90 ul of H₂ O and gentlyagitated for about 10 minutes at 37° C. Subsequently, 10 ul of 9% NaClwas admixed to the surfactant containing solution.

Method B was accomplished by first placing 100 ul of chloroformcontaining 400 ug of phospholipids in a glass tube and removing thechloroform by drying under N₂ for about 10 minutes at 37° C. Sixteen ulof peptide/solvent admixture and 74 ul H₂ O were admixed with the driedphospholipids, and then gently agitated for about 10 minutes at 37° C.To the synthetic surfactant thus formed was admixed 10 ul of 9% NaCl.

Method C was accomplished by first maintaining the polypeptide-PLadmixture at 43° C. for 10 minutes, after which time the solvents wereremoved from the admixture by drying under N₂. When needed, admixtureswere further dried by 15 minutes exposure to vacuum to form a driedpolypeptide-PL admixture. Water was then admixed with each driedadmixture in an amount calculated to equal 90% of the volume necessaryto give a final PL concentration of either 4 or 10 mg/ml (as indicatedin Table 7) to form a second admixture. This second admixture wasmaintained for one hour at 43° C. with agitation. Subsequently, a volumeof 6% NaCl equal to 10% of the volume necessary to give the desired PLconcentration was admixed with the second admixture and the resultingfinal admixture was maintained for 10 minutes at 43° C. In most cases,the final admixture was subjected to a last step of 3 cycles of freezingand thawing.

B. Results

The synthetic surfactants illustrated in Table 6 were prepared asindicated in the table.

                  TABLE 6                                                         ______________________________________                                                                     (2)     (3)                                                                   Phos-   Composite                                                   (1)       pholipid                                                                              Hydro-                                                      Admixture Admixture                                                                             phobicity                                Peptide                                                                             Solvent      Formed    Method  Value                                    ______________________________________                                        p1-15 n-propyl alcohol                                                                           suspension                                                                              A       -16.7                                    p11-25                                                                              H.sub.2 O    solution  B       +1.7                                     p21-35                                                                              Chloroform   suspension                                                                              A       -9.2                                     p31-45                                                                              H.sub.2 O    solution  B       -9.9                                     p41-55                                                                              H.sub.2 O    solution  B       -5.4                                     p51-65                                                                              H.sub.2 O    suspension                                                                              B       -2.2                                     p61-75                                                                              methanol     suspension                                                                              A       -9.9                                     p71-81                                                                              H.sub.2 O    suspension                                                                              B       +3.9                                     p74-81                                                                              H.sub.2 O    solution  B       +3.7                                     p66-81                                                                              methanol:H.sub.2 O                                                                         suspension                                                                              A       -1.0                                     p52-81                                                                              methanol:H.sub.2 O                                                                         suspension                                                                              A       -6.2                                     ______________________________________                                         (1) Each polypeptide was admixed with the indicated solvent to achieve a      concentration of 2.5 ug of peptide per ul of solvent.                         (2) The letters indicate the synthetic surfactant preparation method used     Those methods are described above.                                            (3) The composite hydrophobicity value of each peptide was determined as      described above.                                                         

Each of the synthetic surfactants indicated in Table 6 were assayed forsurfactant activity as evidenced by their ability to reduce surfacetension in vitro using the "bubble assay" of Enhorning as describedabove.

The results of this study, shown in FIG. 6, indicate that a subjectpolypeptide, when admixed with pharmaceutically acceptablephospholipids, forms a synthetic pulmonary surfactant that has greatersurfactant activity than the phospholipids alone, as evidenced by thelower ΔP values. Typically 10% to 80% lower ΔP values were obtainedusing the polypeptides. It should be noted that the 8 amino acid residuecontrol peptide p74-81, which does not conform to the teachings of thepresent invention, did not form a synthetic PS having a greater activitythan the phospholipid alone, thus indicating that amino acid residuelength is a critical feature.

The surfactant activity of additional exemplary polypeptides of thisinvention was studied using the "bubble assay" as described above. Theresults of the study are illustrated below in Table 7.

Each polypeptide was admixed with the indicated solvent at aconcentration of 2.5 mg of polypeptide per ml of solvent. The resultingadmixture was observed to determined whether a solution or a suspensionof insoluble polypeptide was formed. Those admixtures forming asuspension were further admixed by water bath sonication for 10 secondsto form a very fine suspension, sufficient for pipetting using glasspipettes.

After admixture with solvent, each peptide was admixed withphospholipids (PL), DPPC:PG, 3:1, in chloroform in a glass tube so thatthe amount of polypeptide addd equaled one-tenth (10% by weight) of theamount of PL added, to form a synthetic surfactant according to eithermethod A, B or C.

Each of the synthetic surfactants was then assayed for surfactantactivity as evidenced by their ability to reduce surface tension invitro in the bubble assay performed as described above. The pressuregradient (ΔP) is a measure of surfactant activity in the polypeptide-PLfinal admixture which was determined using an Enhorning Surfactometer asdescribed above. Measurements were obtained at time points of 15 seconds(15"), 1 minute (1') and 5 minutes (5') and are expressed as a mean ofthree independent measurements of the indicated polypeptide-PLadmixture. Pressure gradient measurements for comparable samples of PLalone (PL) and natural human surfactants were determined as controls.

The results of this study are shown in Table 7.

                                      TABLE 7                                     __________________________________________________________________________                           (2)                                                                           Phos- (3) (4)                                                        (1)      pholipid                                                                            Conc.                                                                             Pressure                                                   Admixture                                                                              Admixture                                                                           of PL                                                                             Gradient                                     Peptide                                                                            Solvent  Formed   Method                                                                              mg/ml                                                                             15"  1'   5'                                 __________________________________________________________________________    p1-15                                                                              N-propanol                                                                             suspension                                                                             A      4  0.94 0.82 0.48                               p36-81                                                                             50% chloroform                                                                         suspension                                                                             C+    10  0.90 0.87 0.79                                    50% methanol                                                             p44-80                                                                             68% chloroform                                                                         solution C-    .sup. 10.sup.5                                                                    0.77 0.64 0.24                                    32% methanol                                                             p46-76                                                                             64% chloroform                                                                         solution C+    10  0.90 0.80 0.62                                    36% methanol                                                             p51-72                                                                             75% chloroform                                                                         suspension                                                                             C+    10  1.15 0.76 0.33                                    25% methanol                                                             p51-76                                                                             37% chloroform                                                                         solution C+    10  0.99 0.91 0.42                                    63% methanol                                                             p51-80                                                                             45% chloroform                                                                         solution C+    10  0.92 0.89 0.48                                    55% methanol                                                             p51-81                                                                             50% chloroform                                                                         suspension                                                                             C+    10  0.94 0.86 0.64                                    50% methanol                                                             p52-81                                                                             67% DMF  solution A      4  1.33 1.19 0.96                                    33% chloroform                                                           p54-72                                                                             76% chloroform                                                                         suspension                                                                             C+    10  1.28 0.92 0.38                                    24% methanol                                                             p54-76                                                                             71% chloroform                                                                         suspension                                                                             C+    10  0.92 0.82 0.23                                    24% methanol                                                             p59-80                                                                             32% chloroform                                                                         suspension                                                                             C-    .sup. 10.sup.5                                                                    1.10 0.96 0.64                                    68% methanol                                                             p59-81                                                                             68% chloroform                                                                         solution C-     4  1.08 1.02 0.75                                    32% methanol                                                             p64-80                                                                             48% chloroform                                                                         solution C-    .sup. 10.sup.5                                                                    1.07 0.87 0.11                                    52% methanol                                                             p66-81                                                                             40% DMF  suspension                                                                             A      4  1.22 1.11 0.84                                    60% chloroform                                                           p74-81                                                                             water    solution B      4  2.37 2.32 2.31                               DL4  47% chloroform                                                                         solution C-     4  2.00 1.80 1.30                               (31 mer)      53% methanol                                                    RL4  32% chloroform                                                                         solution C-     4  0.58 0.65 0.33                                    68% methanol                                                             RL8  19% chloroform                                                                         suspension                                                                             C+    10  0.68 0.69 0.19                                    81% methanol                                                             RRL7 49% chloroform                                                                         solution C-     4  1.65 1.25 1.00                                    51% methanol                                                             RCL-1                                                                              79% chloroform                                                                         suspension                                                                             C+    10  0.50 0.59 0.06                                    21% methanol                                                             RCL-2                                                                              67% chloroform                                                                         suspension                                                                             C+    10  0.00 0.00 0.00                                    33% methanol                                                             RCL-3                                                                              75% chloroform                                                                         suspension                                                                             C+    10  0.55 0.51 0.33                                    25% methanol                                                             PL                     C+    10  >2.50                                                                              >2.50                                                                              2.33                               Natural Human Surfactant     10  1.06 0.81 0.79                               __________________________________________________________________________     (1) Whether the initial admixture of peptide was a solution or a              suspension is indicated.                                                      (2) The letters indicate the synthetic surfactant preparation method used     Those methods are described above. A "+" indicates that the final             admixture was subjected to a last step of 3 cycles of freezing and            thawing. A "-" indicates the step was not performed.                          (3) Concentration ("Conc.") of phospholipid (PL) in the final admixture i     indicated in milligrams PL per milliliter admixture (mg/ml).                  (4) The pressure gradient is a measure of surfactant activity in the          polypeptidePL final admixture as determined using an Enhorning                Surfactometer as described in Example 2. Measurements were obtained at        three points of 15 seconds (15"), 1 minute (1') and 5 minutes (5') and ar     expressed as a mean of 3 independent measurements of the indicated            polypeptidePL admixture. Pressure gradient measurements for comparable        samples of PL alone (PL) and natural human surfactant are als o shown.        .sup.5 These solutions were made at a concentration of 20 mg/ml PL and        were diluted to 10 mg/ml prior to testing.                               

These results indicate that a subject polypeptide, when admixed withpharmaceutically acceptable phospholipids, forms a synthetic pulmonarysurfactant that has a greater surfactant activity than the phospholipidsalone, as demonstrated by the higher volume per given pressure.

Example 3--In Vivo Assessment of Synthetic Surfactant Activity A.Methods

Preparation of Synthetic Surfactants

A subject polypeptide was first admixed with solvent as described inExample 2. The resulting admixture was further admixed with phospholipid(PL) so that the amount of polypeptide added was either 3%, 7% or 10% byweight of the amount of PL added as indicated below. The finalpolypeptide, PL admixture (synthetic surfactant) was formed according tomethod C using the final freeze thaw step as described in detail in the"Preparation of Synthetic Surfactants" section in Example 2, except thatthe final admixture had a concentration of 20 mg phospholipid per ml offinal admixture.

Instillation Protocols

Protocol 1: Fetal rabbits were treated by injection into the trachea ofa 0.1 ml solution that contained either a synthetic surfactant preparedin Example 3A or either 2 mg of native surfactant (NS) prepared asdescribed in Example 1 or 2 mg PL.

Protocol 2: Synthetic surfactant was instilled in rabbit fetal lung byinjection into the trachea from a single syringe of the following threecomponents such that the components exit the syringe in the followingorder: (1) 0.05 ml air; (2) 0.1 ml of a synthetic surfactant prepared inExample 3A or either 2 mg of PL or 2 mg of native surfactant; and (3)0.1 ml air.

Protocol 3: From one syringe, a 0.1 ml aliquot of synthetic surfactantprepared as described in Example 3A (or 2 mg of NS or of PL), wasinstilled into the rabbit trachea as described above, followed byinjection of 0.05 ml lactated Ringer's Solution and 0.2 ml air from asecond syringe.

Protocol 4: From one syringe, 0.1 ml of a synthetic surfactant preparedas described in Example 3A (or 2 mg of NS or of PL), 0.15 ml air, 0.1 mlsaline, and 0.3 ml air were injected into the trachea as describedabove. Two subsequent aliquots of 0.3 ml air were given.

Protocol 5: Fetal rabbits were treated by injection into the tracheafrom a single syringe the following four components such that the fourcomponents exit the syringe upon injection in the order listed: (1) 0.2ml solution that contains either a synthetic surfactant prepared inExample 3A or either 4 mg of native surfactant, or 4 mg PL; (2) a 0.15ml volume of air; (3) a 0.1 ml normal saline solution; and (4) a 0.3 mlvolume of air. The above injection was then repeated 15 minutes afterthe first injection.

Protocol 6 Rabbits were treated as described in Protocol 5, except thattwo subsequent aliquots of 0.3 ml air were give following the initialinstillation and no additional instillation was given at 15 min.

Fetal Rabbit Model for Studying Surfactant Activity

The surfactant activity of exemplary polypeptides of this invention wasstudied using the methods described in detail previously by Revak, etal., Am. Rev. Respir. Dis., 134:1258-1265 (1986), with the exceptionsnoted hereinbelow.

Twenty-seven day gestation fetal rabbits were delivered by hysterotomyand immediately injected with 0.05 ml Norcuron (Organon, Inc., N.J.) toprevent spontaneous breathing. The fetal rabbits were then weighed and asmall cannula was inserted into the trachea by tracheotomy. Syntheticsurfactant prepared as described above was then instilled into the fetalrabbit lung by one of the above instillation protocols.

Following instillation the rabbit was placed in a specially designedplethysmograph (containing a Celesco transducer) connected to aventilator (Baby Bird, Baby Bird Corp., Palm Springs, Calif.) and theinstilled lung was ventilated at a rate of 30 cycles per minute with apeak inspiratory pressure of 25 cm H₂ O, a positive end expiratorypressure of 4 cm H₂ O and an inspiratory time of 0.5 seconds. In somestudies, dynamic compliance measurements were made at various timesthroughout the ventilation procedure. In others, static compliancemeasurements were made following ventilation.

Static compliance measurements were made after 30 minutes ofventilation. The animals were removed from the ventilator and the lungswere degassed at -20 cm H₂ O in a bell jar under vacuum. Thereafter, thelungs were first inflated and then deflated through a T-connectorattached to a tracheostomy tube. The volume of air required to reachstatic pressures of 5, 10, 15, 20, 25 and 30 cm H₂ O was measured duringboth inflation and deflation phases to generate static compliance.

Using the plethysmograph, dynamic compliance measurements were made byvarious times throughout a 60 minute ventilation period.Computer-assisted data analysis resulted in compliance data expressed asml of air per cm H₂ O per gram of body weight at each time point.Compliance was calculated by the formula below.

    ______________________________________                                        Compliance = ΔV                                                                     ΔP                                                          ______________________________________                                        ΔP.sub.tp =                                                                           (C).sup.-1 · (ΔV) + (R) · (F)           P.sub.tp =    transpulmonary pressure                                         C =           compliance (elastic                                                           component - relates change                                                    in volume to pressure)                                          R =           resistance (relates flow to                                                   pressure)                                                       F =           flow                                                            V =           volume = the integral of flow                                                 with respect to time                                            ______________________________________                                    

The above equation was solved with a multiple linear regression for Cand R. The compliance (C) represents the elastic nature of the lungs andthe resistance (R) represents the pressure necessary to overcome theresistance to the flow of gas into and out of the lungs.

B. Results

Static compliance data using instillation protocols 1 and 5 are shown inFIGS. 7 and 8, respectively. Improved lung compliance was seen in alllungs treated with natural surfactant or with the synthetic surfactantstested as compared with those lungs treated with phospholipids (PL)alone, with one exception. The synthetic surfactant prepared using p1-15(FIG. 7) did not produce improved lung compliance over PL alone whenmeasured by static compliance.

The results of the dynamic compliance studies are illustrated in Table8.

                  TABLE 8                                                         ______________________________________                                        Dynamic Compliance                                                            in ml air/cm H.sub.2 O × 10.sup.6                                       g body weight                                                                 Peptide Minutes after          Sample                                         Compared                                                                              Surfactant Instillation                                                                              Given By                                       To PL   10     20     30   40   50    60   Protocol #                         ______________________________________                                        PL       7      8      7   10   11    15   4                                          24     22     23   23   22    20   4                                          15     16     17   18   21    29   4                                  NS      265    251    168  186  173   147* 4                                          418    388    405  288  237  *     4                                          155    176    172       172  179   4                                  p36-81                                                                         5%                   255             146* 3                                   5%                   245            291   3                                  10%                   154            1,162 2                                  10%                   252            623   2                                  p44-80                                                                        10%.sup.1                                                                             27     87     138  207  323        6                                  10%.sup.1                                                                             20     23     35   59   87   136   6                                  p51-80                                                                        10%.sup.1                                                                             42     114    247  300             6                                  10%.sup.1                                  6                                  p52-81                                                                         5%                   517             226* 3                                   5%                   434             55*  3                                  10%                   195            1,243 2                                  10%                   43             1,690 2                                  p51-76                                                                        10%     33     22     56   87   124   85   4                                  10%     10     11     186  358  141   144* 4                                  10%     15     36     109  241  264  301   4                                  p51-80                                                                        10%     17     41     52   78   99   208   4                                  10%     76     94     149  149  217  308   4                                  10%     23     71     130  156  182   109* 4                                  10%     42     114    247  388             6                                  p59-80                                                                        10%.sup.1                                                                             24     34     68   107  146  132   6                                  10%.sup.1                                                                             55     111    190  300  376        6                                  p64-80                                                                        10%.sup.1                                                                             63     129    235  318             6                                  ______________________________________                                         .sup.1 Prior to instillation into the rabbits, these samples were filtere     through a 25 micron filter.                                                   .sup.* A decrease in compliance with time may indicate the development of     pneumothorax.                                                            

As shown in Table 8, each of the synthetic surfactants of this inventionand natural surfactant improved dynamic compliance values in comparisonto phospholipid alone.

C. Discussion

The in vivo compliance studies demonstrate that the use of a number ofexemplary synthetic surfactants of this invention resulted in enhancedcompliance in comparison to phospholipid alone for each of the assayedsynthetic surfactants. Thus, the proteins and polypeptides of thisinvention when admixed with pharmaceutically acceptable phospholipidsform synthetic surfactants that have greater surfactant activity thanphospholipid alone. Use of the synthetic surfactants is advantageous inproducing improved compliance values in vivo.

Example 4--Study of Binding of C-Terminal Peptide to Lung EpithelialCells A. Methods

Peptide Binding Assay

A peptide having residues 74-80 of SP18 (VLRCSMD) was radiolabeled bythe Bolton-Hunter method (Bolton et al., Biochem J., 133:529-538 1973)with ¹²⁵ I (New England Nuclear--34.1 moles/ml, 28.0 ng/ml, 75 μCi/ml).

Human pulmonary epithelial cells (human lung carcinoma cell, ATCCreference no. CCL 185, commonly known as A549 cells) were grown toconfluence in 6 well tissue culture dishes. The following solutions wereused in this study:

    ______________________________________                                        PBS/BSA:    10 mM Na Phosphate + .15M NaCl +                                              0.5% BSA pH 7.4                                                   Lysis Buffer:                                                                             1% SDS in water                                                   Solution F: 5 ml PBS/BSA + 51.56 μg cold                                               peptide                                                           Solution D: 2.5 ml PBS/BSA + 87 μl .sup.125 I-peptide                      Solution D1/5:                                                                            0.5 ml D + 2.0 ml PBS/BSA                                         Solution D/125:                                                                           0.5 ml D1/5 + 2.0 ml PBS/BSA                                      Solution E: 2.5 ml PBS/BSA + 87 μl .sup.125 I-peptide +                                20.78 μg cold peptide                                          Solution E1/5:                                                                            0.5 ml E + 2.0 ml PBS/BSA                                         Solution E 1/25:                                                                          0.5 ml E1/5 + 2.0 ml PBS/BSA                                      ______________________________________                                    

Three 6 well plates were pretreated by incubating with 0.5 ml of thefollowing solutions for 15 min. at 22° C. The odd-numbered wells werepretreated with PBS/BSA and the even-numbered wells with solution F.Following removal of the pretreatment solution, the wells were incubatedwith 0.5 ml of the following solutions at 22° C. for the indicated timewhile gently rocking the plates.

    ______________________________________                                        Well         Sample      Incubation Time                                      ______________________________________                                        1            D            7 minutes                                           2            E            7 minutes                                           3            D 1/5        7 minutes                                           4            E 1/5        7 minutes                                           5            D 1/25       7 minutes                                           6            E 1/25       7 minutes                                           7            D            30 minutes                                          8            E            30 minutes                                          9            D 1/5        30 minutes                                          10           E 1/5        30 minutes                                          11           D 1/25       30 minutes                                          12           E 1/25       30 minutes                                          13           D           143 minutes                                          14           E           143 minutes                                          15           D 1/5       143 minutes                                          16           E 1/5       143 minutes                                          17           D 1/25      143 minutes                                          18           E 1/25      143 minutes                                          ______________________________________                                    

At the end of the incubation time the supernatant was removed from eachwell and saved for counting. Each well was washed four times with cold(4° C.) PBS/BSA. The washes were saved for counting. The plate was thenbrought back to room temperature and 1 ml of lysis buffer was added toeach well. The plate was gently shaken until all cells had lysed andcome off the plate (3-4 minutes). The solution was removed from eachwell and counted. A second ml of lysis buffer was added to each well,mixed a few minutes and removed for counting of bound counts. Thepercent and absolute amounts of counts were determined.

Specific counts bound were determined by subtracting the counts bound inwells containing unlabeled (cold) peptide from the corresponding wellwithout cold peptide. The results are illustrated in Table 9, below.

The procedure was repeated with the following changes:

D₁ =1433 μl PBS/BSA+167 μl ¹²⁵ I-peptide [1.78 pmol/500 μl]

D₂ =183.3 μl PBS/BSA+366.7 μl D₁ [1.19 pmol/500 μl]

D₃ =275 μl PBS/BSA+275 μl D₁ [0.89 pmol/500 μl]

D₄ =366.7 μl PBS/BSA+183.3 μl D₁ [0.59 pmol/500 μl]

D₅ =458.3 μl PBS/BSA+91.7 μl D₁ [0.30 pmol/500 μl]

D₆ =513.3 μl PBS/BSA+36.7 μl D₁ [0.12 Pmol/500 μl]

E₁ =1386.24 μl PBS/BSA+167 μl ¹²⁵ I-peptide [4.676 ng]+46.76 μl coldpeptide at 100 μg/ml [4.676 μg]

E₂ -E₆ =Diluted as above for D₂ -D₆.

F=3.398 ml PBS/BSA+102.29 μl cold peptide at 100 μg/ml

Two six-well plates were washed once with 1 ml PBS/BSA. The oddnumbered-wells were pretreated with PBS/BSA and the even-numbered wellswith solution F. Following removal of the pretreatment solution, thefollowing solutions were added.

    ______________________________________                                        Well     Sample         Well   Sample                                         ______________________________________                                        1        D.sub.1         7     D.sub.4                                        2        E.sub.1         8     E.sub.4                                        3        D.sub.2         9     D.sub.5                                        4        E.sub.2        10     E.sub.5                                        5        D.sub.3        11     D.sub.6                                        6        E.sub.3        12     E.sub.6                                        ______________________________________                                    

The solutions were incubated for 30 minutes at room temperature withgentle rocking. The supernatants were than removed and saved forcounting. Each well was washed 4 times with 0.5 ml of cold PBS/BSA.Washes were saved for counting. 1 ml of 1% SDA was added to each well tosolubilize the cells. After 3 minutes all the cells could be seen tohave come off the plate. The lysed cell-containing supernatant wascounted, together with a second SDS wash of the wells. Total counts andthe percentage of counts bound were determined. Specific binding wasdetermined by subtracting the counts bound in wells containing coldpeptide from the corresponding well without cold peptide. The resultsare illustrated in Table 10.

B. Results

The results of the binding studies are illustrated below in Tables 9 and10.

                  TABLE 9                                                         ______________________________________                                                        Total                                                                         CPM     %              Specific                               Well  Total CPM Bound   Bound  Difference                                                                            Cts Bound*                             ______________________________________                                        1     1,109,126 24,414  2.23                                                  2     1,087,659 17,353  1.60   0.63%   6,930                                  3       223,170  4,479  2.01                                                  4       221,608  4,113  1.86   0.15%   330                                    5       45,877    828   1.80                                                  6       47,731    880   1.84   -0.04%  -18                                    7     1,103,606 25,905  2.35                                                  8     1,152,287 19,230  1.67   0.68%   7,480                                  9       227,396  4,996  2.20                                                  10      230,974  4,137  1.79   0.41%   901                                    11      47,899   1,030  2.15                                                  12      49,894    922   1.85   0.30%   132                                    13    1,151,347 10,071   .87                                                  14    1,108,755  9,506   .86   0.01%   110                                    15      220,340  1,692   .77                                                  16      229,253  1,800   .79   -0.02%  -44                                    17      46,893    407    .87                                                  18      47,426    386    .81   0.06%    26                                    ______________________________________                                         *Corrected to 1,100,000 cpm/undiluted tube.                              

                  TABLE 10                                                        ______________________________________                                                        CPM      %     Corrected                                      Well  Total CPM Bound    Bound Difference*                                                                            p mol                                 ______________________________________                                        1     3,070,705 66,954   2.78                                                 2     2,995,775 56,055   1.87  9,390    1.78                                  3     2,029,323 39,562   1.95                                                 4     2,013,557 33,573   1.67  5,723    1.19                                  5     1,436,189 26,883   1.87                                                 6     1,427,731 25,073   1.76  1,755    .89                                   7       994,288 15,669   1.58                                                 8       964,481 14,776   1.53    503    .59                                   9       460,317  6,513   1.41   -52     .30                                   10      479,746  6,816   1.42   -52     .30                                   11      202,494  2,930   1.45                                                 12      192,990  2,806   1.45     0     .12                                   ______________________________________                                         *Corrected for 1.78 p mol = 3,033,740 cpm                                

C. Discussion

As can be seen from the data in Table 9, the study demonstrated that thepeptide was binding specifically to the cells as demonstrated bycompetitive inhibition by unlabeled peptide. However, the cells were notsaturated by the amount of labeled peptide used in this study.Additionally, degradation of the peptide was occurring by 143 minutes.

The second study was performed using the 30 minute incubation period andan increased amount of labeled peptide to achieve saturation of thecells. As seen in Table 10, specific binding was again demonstrated.Further, saturation was achieved as demonstrated by leveling-off of theamount of bound counts at high concentration of labeled peptide.

The binding studies thus demonstrate that the C-terminal peptide of thisinvention binds specifically to lung epithelial cells.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the present invention and isnot to be taken as limiting. Numerous other variations and modificationscan be effected without departing from the true spirit and scope of thepresent invention.

What is claimed is:
 1. A polypeptide having an amino acid residue sequence selected from the group consisting of:DLLLLDLLLLDLLLLDLLLLD, RLLLLRLLLLRLLLLRLLLLR, RLLLLLLLLRLLLLLLLLRLL, RRLLLLLLLRRLLLLLLLRRL, RLLLLCLLLRLLLLLCLLLR, RLLLLLCLLLRLLLLCLLLRLL, and RLLLLCLLLRLLLLCLLLRLLLLCLLLR.
 2. A synthetic pulmonary surfactant comprising a pharmaceutically acceptable phospholipid admixed with a polypeptide having an amino acid residue sequence selected from the group consisting of:DLLLLDLLLLDLLLLDLLLLD, RLLLLRLLLLRLLLLRLLLLR, RLLLLLLLLRLLLLLLLLRLL, RRLLLLLLLRRLLLLLLLRRL, RLLLLCLLLRLLLLLCLLLR, RLLLLLCLLLRLLLLCLLLRLL, and RLLLLCLLLRLLLLCLLLRLLLLCLLLR,said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.
 3. A method of treating respiratory distress syndrome comprising administering a therapeutically effective amount of a synthetic pulmonary surfactant, said surfactant comprising a pharmaceutically acceptable phospholipid admixed with an effective amount of a polypeptide having an amino acid residue sequence selected from the group consisting of:DLLLLDLLLLDLLLLDLLLLD, RLLLLRLLLLRLLLLRLLLLR, RLLLLLLLLRLLLLLLLLRLL, RRLLLLLLLRRLLLLLLLRRL, RLLLLCLLLRLLLLLCLLLR, RLLLLLCLLLRLLLLCLLLRLL, and RLLLLCLLLRLLLLCLLLRLLLLCLLLR,said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.
 4. A polypeptide having an amino acid residue sequence represented by the formula: RLLLLRLLLLRLLLLRLLLLR, said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.
 5. A synthetic pulmonary surfactant comprising a pharmaceutically acceptable phospholipid admixed with polypeptide wherein said polypeptide has an amino acid residue sequence represented by the formula: RLLLLRLLLLRLLLLRLLLLR, said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone.
 6. A method of treating respiratory distress syndrome comprising administering a therapeutically effective amount of a synthetic pulmonary surfactant, said surfactant comprising a pharmaceutically acceptable phospholipid admixed with an effective amount of polypeptide wherein said polypeptide has an amino acid residue sequence represented by the formula: RLLLLRLLLLRLLLLRLLLLR, said polypeptide, when admixed with a pharmaceutically acceptable phospholipid, forming a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone. 